【作者】 杨欢欢；

【导师】 刘子龙；

【作者基本信息】 华中科技大学 ， 法医临床与精神病学， 2022， 硕士

【摘要】 背景与目的:近年来,在法医毒理学及毒物分析鉴定中,利用毛发来检测重金属、毒品及药物滥用等方面的研究越来越广泛。与血液、尿液等传统基质相比,毛发和指(趾)甲等角化基质检测滥用药物具有检测窗口宽、获取检材方便、无创、便于储存和运输、检测时间窗长等独特优势。精神障碍患者大多长期服用利培酮、奥氮平和氯氮平等抗精神病药物,其体内代谢与分布特征与部分滥用药物类似。通过对2010年以来发表的利用角化基质检测抗精神病药物的研究分析,发现该领域的研究目前仍处于初步阶段:相关研究相对较少,大多数仅涉及一种或者少量几种抗精神病药物;多数研究是利用标准品建立检测方法,再应用于单个或几个实际样本中进行验证,缺乏剂量等相关的临床信息及系统性研究。本研究尝试借鉴并优化药物滥用的检测方法,利用精神障碍患者的毛发、指甲检材对临床常见的十种抗精神病药物进行定性及定量检测,推测精神障碍患者服用药物的时间窗及角化基质中药物浓度与血药浓度之间的关系,以期为法医精神病鉴定中判断精神病人发生危害行为(如伤人、杀人)或羁押前是否服用抗精神病药物、服药种类、剂量、用药时间及病情严重程度提供有力证据。方法:本研究探索并优化利用超高效液相色谱-四级杆飞行时间液质联用系统(Ultra High Performance Liquid Chromatography-Quadrupole-Time of Flight-Tandem Mass Spectrometry,UHPLC-Q-TOF-MS/MS)同时检测人类毛发、指甲、血液三种基质中常用的十种抗精神病药物(奥氮平、氯丙嗪、氯氮平、奋乃静、舒必利、氟哌啶醇、氨磺必利、喹硫平、利培酮、阿立哌唑)的检测方法,并应用于54名临床上接受固定剂量药物治疗的精神分裂患者提供的血液、毛发和指甲样本及2例本中心受理的精神病患者杀人鉴定案件的精神鉴定中。研究中血液样本采用乙腈蛋白沉淀法进行处理,毛发、指甲样本用甲醇、水、甲醇依次洗涤,液氮研磨仪粉碎,再置于甲醇溶液中38℃浸提18小时,蛋白沉淀液及浸提液经MEMBRANA滤膜(0.22um)过滤后置于UHPLC-Q-TOF-MS/MS系统中分析。色谱分析系统使用Agilent Poroshell 120 EC-C18 2.7 um(3.0*150 mm)色谱柱;流动相为5m M乙酸铵/0.1%甲酸水溶液和乙腈;质谱系统采用电喷雾离子源,正离子模式,多重离子监测(Multiple Reaction Monitoring MRM)进行测定。结果:(1)该检测方法中所有分析物在色谱图峰形均表现良好且无内源性及外源性干扰;定量限在血液中为0.01~0.005ng/ml、毛发中为0.01~0.005ng/mg、指甲中为0.005ng/mg;准确度在不同基质中分别为(血液:96.0%~101.6%;毛发:97.5%~102.5%;指甲:97.6%~101.7%);各分析物在不同基质中精密度相对标准偏差(Relative Standard Deviation RSD)均低于5%;提取率为:血液:78.1%~107.4%,毛发:73.9%~93.5%,指甲:75.2%~90.5%;基质效应为血液:-36.5%~17.1%,毛发为-20.6%~8.9%,指甲为-24.3%~11.3%;样本在进样器72h内的稳定性:血液:91.4%~99.8%;毛发:88.1%~105.3%;指甲:86.1%~102.6%。(2)样本检测结果:本研究对54名临床上接受固定剂量药物治疗患者的血液、毛发和指甲样本进行了分析,并对10例女性患者提供的较长的头发样本(6~20cm)进行了分段检测。研究发现,所有药物的血药浓度检测范围均在AGNP精神科治疗药物监测共识指南推荐的药物范围内,毛发药物浓度范围与已发表文献中的数据中较为相似,而指甲中抗精神病药物浓度研究较少,缺乏相应的检测数据,部分药物(氯丙嗪、舒必利、氨磺必利、利培酮、阿立哌唑)在指甲中的药物浓度为本次研究中首次报道。(3)对比不同基质中药物浓度与服药剂量之间的关系发现,氯氮平、喹硫平服药剂量与生物基质(血液、毛发、指甲)间具有显著相关性,其中服药剂量与血药浓度间具有极显著相关性(氯氮平:r=0.7901,P<0.0001;喹硫平:r=0.8325,P=0.0054);对比不同基质间药物浓度发现,有五种药物的血液与毛发中药物浓度均有显著相关性(奥氮平:r=0.5924,P=0.0096;氯氮平:r=0.6624,P=0.004;喹硫平:r=0.7178,P=0.0294;利培酮:r=0.6909,P=0.0005;阿立哌唑:r=0.6227,P=0.0408)。(4)毛发样本分段检测发现,除奥氮平在毛发中性质不稳定,在毛发第3~4分段后就无法检测出来外,其余五种药物(氯氮平、喹硫平、阿立哌唑、舒必利、利培酮)在毛发检材中较为稳定,任意毛发分段中均能检测到药物存在,尤其是接受阿立哌唑治疗的患者,停药3~6个月后,仍能在毛发阶段中检测出相应药物。(5)对实际案件中曾服用精神药物的被鉴定人提供的毛发及指甲样本进行检测发现,均检出了利培酮成分,经警方证实,检出药物与患者此前服用药物一致。结论:(1)本研究中优化的检测体系在标准品及实际样本检测中均具有较好的选择性和稳定性,能够准确区分多种被测抗精神病药物。(2)奥氮平、氯氮平、阿立哌唑、喹硫平、利培酮的血药浓度与毛发浓度具有显著相关性,可以将毛发作为临床上监测抗精神病药物浓度的补充方案。(3)毛发分段检测发现,不同药物在毛发中具有不同的分布特征,固定剂量下,药物浓度表现为发根到发尾逐渐降低或者而基本不变。(4)在鉴定实际案例的初步应用表明,该方法在推断精神病人发生危害行为或羁押前是否服用抗精神病药物及服药种类具有可行性。更多还原

【Abstract】 Background: In recent years,the use of hair to detect heavy metals and drug abuse has been increaseding significantly in the field of forensic toxicology and toxicological analysis.Compared with traditional matrices(e.g.,blood,urine)such as hematuria,hair and fingers(toes),keratinizing matrices including hair and fingers(toes)have unique advantages such as wide detection window,convenient material acquisition,non-invasive,convenient storage and transportation,and long detection time window.Given that patients with mental disorders often require long-term use of antipsychotics,such as risperidone,olanzapine,and clozapine,their metabolic and distribution characteristics of the drugs in these patients are similar to those of some with drug s of abuse.A review of the literature on using matrix to detect antipsychotics since 2010 shows that this trialthis field is still in its preliminary stage: there are relatively only few studies and applications,most of which involved only one or a small number of drugs.In addition,most existing studies used standard substances to establish detection methods and then apply applied them to a single or multiple actual samples for verification,lacking dose and other relevant clinical information(e.g.,dosage)and systematic researchinvestigation.In this study,we validated a method based on detection method for drug abuse to detect antipsychotic drugs in patients with mental disorders using hair and nail samples,in order to provide objective evidence for judging whether patients with mental disorders had taken antipsychotic drugs before prior to the occurrence of violent attack(e.g.,hurting,killing)wounding,killing or detention.And toand inferring the type,dose and duration of the identified person taking antipsychotic drugs,as well as the type and severity of the mental disorders according to the sample test results.Methods: In this study,we explored and optimized the method of detection of commonly used antipsychotic drugs(olanzapine,chlorpromazine,clozapine,perphenazine,sulpiride,haloperidol,amisulpride,quetiapine,risperidone,aripiprazole)using human hair,nail and blood simultaneously via application of Ultra High Performance Liquid Chromatography-Quadrupole-Time of Flight-Tandem Mass Spectrometry(UHPLC-Q-TOF-MS/MS)to simultaneously detected the pathway system of commonly used antipsychotic drugs(olanzapine,chlorpromazine,clozapine,perphenazine,sulpiride,haloperidol,amisulpride,quetiapine,risperidone,aripiprazole)using human hair,nail and blood.And apply applied the method to the blood,hair and nail samples from 54 patients who received fixed doses of antipsychotic drugs in clinical settings,and 2 cases subjects of who were under the psychiatric evaluation of mentalafter homicide in our forensic setting.In this study,blood samples were treated by acetonitrile protein precipitation method,hair and fingernail samples were washed with methanol,water and methanol in turn,then smashed with liquid nitrogen lapping machine and placed in methanol solution at 38℃ for 18 h.The protein precipitate and extract were filtered by 0.22 um membrane and analyzed in UHPLC-Q-TOF-MS/MS system.Chromatographic analysis system using Agilent Poroshell 120 EC-C18 2.7um(3.0 * 150 mm)column;The mobile phase consisted of 5m M ammonium acetate / 0.1% formic acid aqueous solution and acetonitrile.Multiple Reaction Monitoring(MRM)was used to determine the positive ion mode of electrospray ion source.Results:(1)The chromatographic peak shapes of all analytes in this method were performed well and there was no endogenous or exogenous interference;The limits of quantitation were 0.01-0.005 ng/ml in blood,0.01-0.005 ng/mg in hair and 0.005ng/mg in fingernails.The accuracy in different substrates was(blood: 96.0% ~ 101.6%;Hair: 97.5% ~ 102.5%;Nails: 97.6% ~ 101.7%);The Relative Standard Deviation(RSD)of precision of each analyte in different substrates was lower than 5%.Extraction rate: blood: 78.1% ~ 107.4%,hair: 73.9% ~ 93.5%,fingernails: 75.2% ~ 90.5%;Matrix effect was-36.5%-17.1% in blood,-20.6%-8.9% in hair,and-24.3%-11.3% in nails.Stability of samples within 72 h of injector: blood: 91.4% ~ 99.8%;Hair: 88.1% ~ 105.3%;Nails: 86.1% ~ 102.6%.(2)Sample test results: In this study,blood,hair and nail samples from 54 patients who received clinical fixed-dose drug therapy were analyzed,and longer hair samples(6-20cm)provided by 10 female patients were segmented.The study found that the detection range of blood drug concentration of all drugs was within the recommended range of AGNP psychiatric drug surveillance consensus guidelines,and the concentration range of drug concentration in hair was similar to the data in the published literature,while the concentration of antipsychotic drugs in fingernails was rarely studied and corresponding detection data were lacking.The concentrations of some drugs(chlorpromazine,sulpiride,aminosulpiride,risperidone and aripiprazole)in fingernails were reported for the first time in this study.(3)By comparing the relationship between drug concentration and dose in different substrates,it was found that there was significant correlation between clozapine and quetiapine dose and biological matrix(blood,hair,fingernail),among which there was extremely significant correlation between drug dose and blood concentration(clozapine: r =0.7901,P <0.0001;Quetiapine: r=0.8325,P=0.0054);Comparison of drug concentrations between different substrates showed that there was a significant correlation between drug concentrations in blood and hair of five drugs(olanzapine: r=0.5924,P=0.0096;Clozapine: r=0.6624,P=0.004;Quetiapine: r=0.7178,P=0.0294;Risperidone: r=0.6909,P=0.0005;Aripiprazole: r=0.6227,P=0.0408).(4)Hair samples segment detection: in addition to the unstable nature of olanzapine in the hair,the hair section cannot be detected after 3 ~ 4,the rest of the five kinds of drugs(clozapine,quetiapine,sulpiride,aripiprazole and risperidone)is relatively stable in the hair for examination,and was able to detect drugs exist any hair section,especially the patient who receive aripaiprazole treatment,even if 3 ~ 6 months after drug withdrawal,the corresponding drugs can still be detected in the hair stage.(5)It was found that risperidone was found in the hair and nail samples provided by the identified persons who had taken psychotropic drugs in the actual case.The police confirmed that the detected drugs were consistent with the drugs the patient had taken before.Conclusions:(1)the optimized detection system in this study has good selectivity and stability in the detection of standard substances and actual samples,and can accurately distinguish a variety of tested antipsychotic drugs.(2)Blood concentrations of olanzapine,clozapine,aripiprazole,quetiapine and risperidone were significantly correlated with hair concentration,so hair could be used as a supplement for clinical monitoring of antipsychotic drug concentration.(3)Hair segmentation detection showed that different drugs had different distribution characteristics in hairat fixed dose: drug concentration decreased gradually from hair root to hair end or remained basically unchanged.(4)The preliminary application in the identification of actual cases shows that this method is feasible to infer whether or not psychiatric patients take antipsychotic drugs and the types of antipsychotic drugs before the occurrence of harmful behaviors or custody.更多还原